The present invention relates in one aspect to acidified nitrite as an antimicrobial agent, and to a complex of nitrogen oxides arising from the interaction of nitrite and acid as an antiviral composition for the treatment of viral diseases of the skin by topical application thereto. Such nitrogen oxides include in particular NO which is of importance particularly if acidified.
An active entero-salivary circulation in man provides a continuous flow of nitrate into the mouth where it is rapidly reduced to nitrite by bacteria on the tongue. The effect of salivary nitrate excretion is to provide a precursor for the generation of nitrogen oxides by the break down of the nitrite.
In brief we have found that exposure of a yeast, Candida albicans and the bacterium E coli to concentrations of nitrite in saliva together with acid conditions similar to those found in the stomach for one hour caused a dose-dependent reduction in their survival. It is apparent therefore that the generation of nitrogen oxides and/or nitrous acid in the mouth and in the gastrointestinal tract, particularly the upper gastrointestinal tract, from acidified nitrite is preventative of microbial infection.
In the mouth bacteria rapidly reduce nitrates to nitrites. Once swallowed the acid conditions of the stomach protonate the nitrite to form nitrous acid (pKa approx 3.5). The nitrous acid in turn dissociates to form oxides of nitrogen as shown below.
NO2xe2x88x92+H+=HNO2xe2x80x83xe2x80x83(1) 
2HNO2=H2O+N2O3xe2x80x83xe2x80x83(2) 
N2O3=NO+NO2xe2x80x83xe2x80x83(3) 
N2O3+C2H8O6=2NO+H2O+C6H6O6xe2x80x83xe2x80x83(4) 
Endogenous and dietary nitrate is actively concentrated by salivary glands to more than 10 times the concentration in plasma and secreted in saliva. Thus the saliva provides a continuous source of nitrate to the upper gastrointestinal tract. Oral conversion of nitrate to nitrite is rapid and is restricted to the surface of the tongue in man and to the posterior third of the tongue in the rat.
The function of the entero-salivary circulation of nitrate is not known but it may well be that gastric acid by itself is not always sufficient to destroy many ingested micro-organisms and that the primary role of salivary nitrate secretion and conversion to nitrite is as a precursor for nitrogen oxides in the lumen of the stomach which will kill swallowed micro-organisms.
The above identified mechanism is also applicable to the destruction of micro-organisms on an in the skin. For example athlete""s foot or tidea pedis.
In WO 95/22335 we have disclosed a pharmaceutical composition comprising a pharmaceutically acceptable source of nitrites and a pharmaceutically acceptable acidifying agent, inter alia for the direct treatment of disease by topical application. These compounds have a direct effect on the organism concerned but the precise mode of action is not known.
U.S. Pat. No. 4,595,591 reveals a composition comprising an aqueous solution of nitric acid and nitrous acid at a pH below 1 preferably with a organic acid and copper and cadmium ions for the treatment of superficial lesion of the skin, for example tumorous growths.
U.S. Pat. No. 5,648,101 provides a vaso-active composition comprising NO adapted for delivery to a body site inter alia by means of a cream or ointment. The NO is generated from an admixture of ferrous sulphate, an organic acid and an inorganic nitrite and caused to be reactive in the presence of moisture adjacent or at the site. Acidification is not discussed.
WO 96/02268 reveals the inhibition of a virus by nitric oxide (NO2) derived from a complex unstable organic molecule, but the advantages of reduction of pH at the environment of use have not been appreciated, neither have the beneficial effects the chemical release of the NO and NO2 moieties immediately adjacent to the environment of use, been realized.
WO 93/25213 reveals a composition comprising nitrous oxide contained in a dermatological composition comprising as an essential feature a fatty acid or a lower alkyl ester thereof, pH values, particularly at the environment of use, are not mentioned.
All are single formulations which are admixed well prior to application to the environment of use so that NO and NO2 all escape prior to use and hence have a very limited utility.
We have now found inter alia that nitrite at concentrations of up to 4% in an inert carrier cream or ointment when mixed with an organic acid such as salicylic acid reacts to produce oxides of nitrogen which are effective in killing infectious organisms on the skin including fungi, yeast, bacteria and viruses. The combination of nitrite and acid causes mild erythema (redness) of the skin due to release of nitric oxides at the environment of use but this causes no significant inflammation.
We have also found that as far as viruses, as opposed to bacteria for example, are concerned, that the above nitrogen oxide complex, comprising for example NO and/or NO2 while it may effect replication to a degree, more importantly modifies the virally infected cells such that the immune system can recognize the viral particles. Inter alia, this is supported by the fact that the complex is less effective in immunosuppressed hosts. Generally the greater the percent of nitric oxide (NO) the better the immuno-potentiation. If possible up to 100% NO can be used.
It is thought, although more work is required, that smaller molecules, particularly NO and NO2 penetrate the skin by direct diffusion or via the seat glands or hair follicles through the epidermis to the sweat cells. It has been found that although the healthy keratinocytes find the oxides of nitrogen toxic they do not die as they are relatively resistant to its effects. However, the surprising clinical results in our examples lead us to believe that virally infected cells are more susceptible to these effects, leading to destruction of the virally infected cells via a combination of toxicity leading to programmed cell death and potentiation of the immune response to the presence of the virus.
The above identified mechanism is also useful in the sterilisation of objects such as dentures by utilising a sterilizing nitrate solution. Conventional solutions which are effective in sterilising dentures often taste unpleasant due to chlorine-based disinfectants. A combination of nitrite and acid results in a antimicrobial solution which has little or no taste. Other objects such as contact lenses may be sterilised in the same way.
Gastroenteritis continues to be a major problem in rearing pigs and other farm animals. Enteropathogenic Escherichia coli (especially those bearing the K88 antigen) are particularly implicated. Although gastric acidity is thought to be one of the main host defence systems which provides a barrier to orally-acquired infection, this is clearly ineffective in preventing organisms from reaching the more distal intestine in these animals.
The role of NO as a compound which inhibits viral replication in vitro has been disclosed by J. B. Mannick; 63rd Forum in Immunology, and papers in Intervirology 1995; 38: 206-213, Trends in Microbiology 1995; 3: 81-82, Science 1993; 261: 1445-1448, and The Journal of Clinical Investigation 1993; 91: 2446-2452. The above papers disclose the effects of NO on various viruses, for example herpes simplex virus, vaccinia virus and vesicular stomatitis virus. Exogenous NO donors such as S-nitroso-N-acetyl penicillamine(SNAP)or SIN-1 were used in vitro to determine the role of NO as an antiviral compound. Application of exogenous NO to cell-lines infected with the virus under test resulted in inhibition of the viral DNA replication. The exact mechanism of the inhibition seemed to differ depending on the virus involved. For example in the case of vaccinia virus it is thought that the NO may inhibit replication by binding to non-haem iron or thiol groups that are essential for the catalytic activity of enzymes involved in vaccinia replication. In this in vitro model the antiviral effects of NO do not require immune recognition of infected cells thus providing an early defence against viral pathogens prior to the development of a specific immune response.
In order for viruses to survive and reproduce they must evade recognition by the hosts immune responses. The mechanism by which this is achieved is largely unknown but an effective immune response eradicates the infection. Viruses are obligate intracellular pathogens. They reproduce using the host""s metabolic machinery.
At present drug treatment of viral diseases is predicted upon a small number of compounds which block the replication of the virus. For example Acyclovir, which is effective against herpes virus, is a deoxyguanosine analogue which competes with deoxyguanosine triphosphate as a substrate for viral thymidine kinase and when phosphorylated and incorporated in the viral DNA causes premature DNA chain termination.
Unfortunately anti-viral drugs are only effective for a limited number of viral infections and viruses can mutate to overcome the effectiveness of the drugs. In the case of molluscum contagiosum 1 and 2, which are related to orthopox and parapox viruses and share some homology with vaccinia, other forms of treatment have to be used. Current therapies comprise physical destruction with manual extrusion, liquid nitrogen therapy or curettage, all of which are painful and not very effective and may cause scarring. The pain of these therapies is particularly pertinent because the majority of patients are under the age of 10 years.
In the case of recalcitrant warts, destructive therapies such as liquid nitrogen can be used in cases where the conventional salicylic acid paints have not resulted in the warts disappearance. One problem with warts is that the viral pool is in the stem cells which are found at the base of the epidermis. The aforementioned treatments often remove the virus particles and thus the infection from the top layer of the epidermis, but they do not penetrate deep enough to remove the stem cells and therefore the origins of the infection. This can result in the re-emergence of the warts.
An alternative treatment for warts is by use of dinitrochlorobenzene. Such treatment is intended to make the patient allergic to dinitrochlorobenzene, whereupon the patient""s immune system mounts an immune response to the dinitrochlorobenzene at the site of the wart and the wart in some cases disappears, presumably as a result of immuno-potentiation. Immuno-potentiation can be an effective treatment but subjecting the patient to an allergic reaction caused by dinitrochlorobenzene can be hazardous, variable and difficult to control.
According therefore to a first aspect of the present invention there is provided a dosage form for the treatment of bacterial, virus, or fungal conditions in the human or animal body which comprises:
a pharmaceutically acceptable acidifying agent in an amount sufficient to reduce the pH at an environment of use to below pH4, and
a pharmaceutically acceptable source of nitrite ions or a nitrate precursor therefor;
wherein said acidifying agent and said source of nitrite ions or nitrate precursor are separately disposed in respective pharmaceutically acceptable carriers for admixture at the intended environment of use to release NO or NO2 ions.
Preferably the acidifying agent is an organic acid, for example salicylic acid or ascorbic acid. The precursor for the NO2 or NO moiety may be an alkaline metal or alkaline earth metal nitrate capable of conversion to NO2 or NO by enzymic action.
The pharmaceutical acceptable carrier or diluent may be an inert cream or ointment. In a particularly preferred form of the invention the acidifying agent and the source of nitrite ions or precursor therefor are separately disposed in said cream or ointment for admixture to release NO2 or NO ions at the environment of use. Alternatively an acid composition may be presented for administration in tablet or liquid form.
Depending on the type of viral infection the components of the nitrogen oxide can work synergistically or alone. Nitrogen oxides, for example NO and NO2, particularly can diffuse through the epidermis. In the case of warts this allows them to reach the stem cells which are at the base of the epidermis and are the cells which contain the pool of established virus. Once at the infected cells the nitrogen oxide complex can facilitate programmed cell death, selectively in infected cells, which may then be taken up by phagocytes and antigen presenting calls leading to immune recognition of the previously hidden viral antigens. Once recognized, specific immunity will lead to destruction of all infected cells through cellular and humoural responses.
Accordingly therefore to a further aspect of this invention there is provided a method of exposing virally infected cells at or adjacent an environment of use to a mammalian immune response in vivo, which comprises applying to said cells an admixture of nitrogen oxides generated by admixing at the environment of use a pharmaceutically acceptable acidifying agent in an amount sufficient to reduce the pH at the environment of use to below pH4 and a pharmaceutically acceptable source of nitrogen oxides or a nitrate precursor therefor.
According to a second aspect of the invention there is provided the use in the manufacture of a topical medicament for the in vivo potentiation of the immune system during a viral skin infection resultant from virus replication in the epidermis, of topical formulations comprising a separately disposed source of pharmaceutically acceptable nitrogen oxides, and a separately disposed pharmaceutically acceptable acidifying agent, for admixture, at an intended environment of use to release NO and NO2 moieties.
Preferably the viruses replicating in the epidermis which cause the viral skin infection are selected from molluscum contagiosum, herpes simplex type 1 and 2, varicella zoster virus and papilloma virus. Treatment using the acidified nitrogen oxide source has been shown to be particularly effective in viral skin infections caused by the aforementioned viruses.
Conveniently the source of nitrogen oxides contains nitric oxide and may also contain NOxe2x88x92 or NO+ nitrosium ions or a precursor therefor produced when a pharmaceutically acceptable acidifying agent and a pharmaceutically acceptable donor of nitrogen oxides or a precursor therefor, are brought into intimate contact at a site of biological action (environment of use).
If the pharmaceutically acceptable acidifying agent and the pharmaceutically acceptable donor of nitrogen oxides, or a precursor therefor were brought into contact before reaching the site of biological action the efficacy of the treatment is diminished as the nitrogen oxides become progressively more inactive with time.
In a preferred embodiment the pharmaceutically acceptable acidifying agent, the pharmaceutically acceptable donor nitrogen oxides or a precursor therefor are each separately disposed in a pharmaceutically acceptable carrier or diluent.
Preferably the pharmaceutically acceptable acidifying agent is an organic acid or salt with a low pH such as ascorbyl palmitate. The organic acid may be selected from at least one of ascorbic acid, ascorbyl palmitate, salicylic acid, lactic acid, citric acid, formic acid, benzoic acid and tartaric acid.
The choice of acidifying agent depends on the type of infection of the skin and the reaction of the infected areas to treatment. The use of reducing acids such as ascorbic acid gives a quick burst of NO and NO2 with significantly more NO produced compared to the amount of NO2 produced. The other organic acids such as salicylic acid give a sustained concentration of NO and NO2 over a certain time period wherein the ratio of NO to NO2 is low. The concentration of the inorganic nitrite, for example sodium nitrite (or other alkali metal nitrites), as the pharmaceutically acceptable donor of nitrogen oxides or a precursor therefor depends on the acid used and the concentration of the acid used. The reducing acid ascorbic acid is highly reactive so therefore only between 1-10% is required with stoichiometric concentrations of the pharmaceutically acceptable donor of nitrogen oxides or a precursor therefor (e.g. sodium or other alkali metal nitrite). Ascorbyl palmitate is more stable but requires a higher concentration than ascorbic acid because the palmitate has a higher molecular weight. A concentration of between 3% and 25% of ascorbyl palmitate is thus required. If salicylic acid is used, concentrations of between 0.5% and 30% are appropriate, citric acid requires a yet higher concentration of up to 45%. (All % given herein are by weight).
The concentration of sodium nitrite required to react with the above mentioned concentrations of organic acid is between 0.5% and 30%, preferably between 5% and 20%. Other pharmaceutically acceptable sources of nitrogen oxides or a precursor therefor require different ranges of concentration.
Preferably the pharmaceutically acceptable acidifying agent and the pharmaceutically acceptable donor of nitrogen oxides or a precursor therefor are in stoichiometric concentrations. If the pharmaceutically acceptable acidifying agent and the pharmaceutically acceptable donor of nitrogen oxides or a precursor therefor are in stoichiometric concentrations, after the reaction is completed there will be no unreacted compounds left. Accordingly any compounds remaining on the infected area will not be able to contaminate healthy skin with the active medicament or anything the treated area touches such as furniture and clothing.
In a preferred embodiment the medicament is in the form of a paint, a varnish, an ointment, a wax, a salve, or a cream. These embodiments allow the pharmaceutically acceptable acidifying agent and a pharmaceutically acceptable donor of nitrogen oxides, or a precursor therefor to be applied directly to the infected area. The treatment comprising the topical application of separate compositions according to this invention is preferably continued for at least one month, and more preferably two months.
In a further aspect of the present invention there is provided a two-part delivery system for the topical application of a medicament for the in vivo treatment of the epidermis, the said system comprising separately;
a first waxy component comprising a pharmaceutically acceptable acidifying agent;
and a second waxy component comprising a pharmaceutically acceptable source of nitrogen oxides whereby if topically applied in vivo simultaneously, or immediately sequentially, to the environment of use, active nitrogen oxides are released therefrom.
In a further embodiment, the first and second waxy components comprise a paraffin. The acidifying agent is preferably a reducing organic acid or salt such as ascorbic acid or ascorbyl palmitate. The source of nitrogen oxides may be an alkali metal nitrite such as sodium nitrite.
The use of a reducing acid or salt thereof results in a product released at the environment of use which comprises a major amount of NO which has significant therapeutic and immunological effects.
Thus the invention provides for the use of a source of oxide(s) of nitrogen in the manufacture of a composition for the treatment or prophylaxis of a viral skin infection by a virus selected from herpes simplex types 1 and 2, varicella zoster, vaccinia or papilloma, and particularly from molluscum contagiosum.
In a further aspect of the invention there is provided a delivery system for the topical application of a medicament for the in vivo treatment of the epidermis, comprising an adhesive layer and a support layer impregnated with at least one of the components of the medicament, characterized in that the components of the medicament are a pharmaceutically acceptable acidifying agent and a pharmaceutically acceptable donor of nitrogen oxides or a precursor therefor, and a means of combining the pharmaceutically acceptable acidifying agent with the donor of nitrogen oxides. For example the delivery system may comprise two layers, which when in situ release the oxides of nitrogen including nitric oxide. The activation can be by pressure applied or by hydration from the skin.
Preferably the delivery system is adapted for the potentiation of the immune system during a viral skin infection resultant from virus replication with the delivery system in place, such a system may, for example, resemble an adhesive plaster so it is then simple to apply physical pressure to the exterior of the plaster.
Conveniently the donor of pharmaceutically acceptable nitrogen oxides may be aqueous and encapsulated in microspheres or liposomes disposed in the support, preferably in the form of a film or a gauze. The film or gauze allows increased concentrations of the pharmaceutically acceptable acidifying agent to be used. If a solution of salicylic acid is used then only a concentration of 20-26% by weight is applied, but if salicylic acid is impregnated in the film or the gauze then a concentration of 26 to 44% by weight can be applied.
A further advantage of using an adhesive layer is that it can be used to occlude the infected area during treatment which increases the concentration of nitrogen oxides being absorbed through the epidermis.
Another advantage of using the delivery system as just described, instead of two creams or ointments, is that the components of the medicament will only be applied to the infected site, i.e. no spillage will occur. It is also easier for the elderly, who may have shaky hands, to apply the adhesive layer rather than applying a paint. For the treatment of molluscum contagiosum, which is mainly found in those under the age of 10 years, the adhesive layer can be a decoratively patterned in order to appeal to children.
As stated above preferably the integrity of the vehicle is disrupted by pressure after application of the adhesive layer and film or gauze to a site of viral infected skin. If the pharmaceutically acceptable acidifying agent and the pharmaceutically acceptable nitrogen oxide donors or precursors therefor are not kept separate until administration at the site of biological action they will react together thus rendering the medicament less effective. Accordingly, in this embodiment it is necessary for the pharmaceutically acceptable acidifying agent and the pharmaceutically acceptable nitrites or precursors therefor to be retained separately within the film or gauze layer. The application to the site of biological action of pressure applied to the adhesive layer, and therefore the film or gauze layer, can result in the vehicles, such as the microspheres or lipsomes, breaking and the pharmaceutically acceptable acidifying agent and the pharmaceutically acceptable nitrogen oxide donors or precursors therefor reacting, thus treating the infected area.
In another aspect the delivery system may be used in conjunction with a topically applied medicament. The topically applied medicament being either a pharmaceutically acceptable acidifying agent or a pharmaceutically acceptable donor of nitrogen oxides or a precursor therefor.
It is thus possible to provide only one of either the pharmaceutically acceptable acidifying agent or the pharmaceutically acceptable nitrogen oxide donors or precursors therefor impregnated in the film or gauze layer. The other compound, which is not impregnated in the film or gauze can then be applied topically to the infected site. The advantage of this arrangement is that the film or gauze layer can be larger than the infected site but a reaction between the pharmaceutically acceptable acidifying agent and the pharmaceutically acceptable nitrogen oxide donors or precursors therefor only occurs at the infected site where the medicament had been topically applied.
It is also possible to vary the treatment regime by changing the topically applied medicament without changing the compound in the delivery system. For example if the pharmaceutically acceptable nitrogen oxide donors or precursors therefor are impregnated in the film or gauze, then the type of pharmaceutically acceptable acidifying agent that is topically applied can be altered and the same adhesive and film or gauze layers utilized.
The delivery system is an ideal form of treatment for the verrucae on the feet because the delivery system is hidden from view.
In a further aspect of the invention there is provided a method of sterilising an object which method comprises the steps of:
1) preparing a pharmaceutically acceptable acidifying agent and a pharmaceutically acceptable source of nitrite ions,
2) mixing said acidifying agent with said source of nitrite ions in a liquid carrier or diluent in contact with said object thereby to reduce the pH to below 4 while causing said sterilant nitrite ions to sterilize said object.
In a further form of the invention there is provided a sterilant composition comprising a pharmaceutically acceptable acidifying agent,
a pharmaceutically acceptable source of nitrite ions or a nitrate precursor therefor,
and a pharmaceutically acceptable carrier or diluent therefor wherein the acidifying agent is adapted to reduce the pH at the environment of use to below pH4.
In a still further form of the invention there is provided an animal feed supplement comprising a pharmaceutically acceptable acidifying agent and,
a pharmaceutically acceptable source of nitrite ions or a nitrate precursor therefor or, in an amount sufficient to produce a beneficial anti-microbial pharmalogical effect, but insufficient to produce adverse action in the target animal.
The acidifying agent may be salicylic or ascorbic acid as above, and the source of nitrite ions or nitrate precursor therefor may be in an inorganic nitrate as set forth above. Where the animal is the pig, the supplement should be included in an amount sufficient to ensure that each adult animal will receive a balanced dose of between 0.3 to 5.0 g/day and preferably about 1 g/day.